Abstract
Cancer progression towards metastasis follows a defined sequence of events described as the metastatic cascade. For extravasation and transendothelial migration metastatic cells interact first with endothelial cells. Yet the role of endothelial cells during the process of metastasis formation and extravasation is still unclear, and the interaction between metastatic and endothelial cells during transendothelial migration is poorly understood. Since tumor cells are well known to express TGF-β, and the compact endothelial layer undergoes a series of changes during metastatic extravasation (cell contact disruption, cytoskeletal reorganization, enhanced contractility), we hypothesized that an EndMT may be necessary for metastatic extravasation. We demonstrate that primary cultured rat brain endothelial cells (BEC) undergo EndMT upon TGF-β1 treatment, characterized by the loss of tight and adherens junction proteins, expression of fibronectin, β1-integrin, calponin and α-smooth muscle actin (SMA). B16/F10 cell line conditioned and activated medium (ACM) had similar effects: claudin-5 down-regulation, fibronectin and SMA expression. Inhibition of TGF-β signaling during B16/F10 ACM stimulation using SB-431542 maintained claudin-5 levels and mitigated fibronectin and SMA expression. B16/F10 ACM stimulation of BECs led to phosphorylation of Smad2 and Smad3. SB-431542 prevented SMA up-regulation upon stimulation of BECs with A2058, MCF-7 and MDA-MB231 ACM as well. Moreover, B16/F10 ACM caused a reduction in transendothelial electrical resistance, enhanced the number of melanoma cells adhering to and transmigrating through the endothelial layer, in a TGF-β-dependent manner. These effects were not confined to BECs: HUVECs showed TGF-β-dependent SMA expression when stimulated with breast cancer cell line ACM. Our results indicate that an EndMT may be necessary for metastatic transendothelial migration, and this transition may be one of the potential mechanisms occurring during the complex phenomenon known as metastatic extravasation.
Highlights
Endothelial-mesenchymal transition (EndMT) is an embryonic program necessary for organ development
To assess whether primary rat brain endothelial cells (BEC) (RBEC) could undergo transition towards a mesenchymal phenotype, rat brain endothelial cells (RBECs) were treated with 10 ng/ml of TGF-β1, and the subsequent changes were examined by immunofluorescence microscopy and Western blot
This peripheral staining disappeared 48 hrs after TGF-β1 treatment and RBECs became negative for claudin-5 and VE-cadherin expression (Fig. 1a)
Summary
Endothelial-mesenchymal transition (EndMT) is an embryonic program necessary for organ development. Despite being normally dormant in adult organisms, this mechanism can be reactivated during several pathological conditions, such as cancer and fibrosis. At cellular and molecular level EndMT is regulated by similar factors and signaling pathways under both physiological and pathological conditions. EndMT was first described during heart development [1]. EndMT contributes to the formation of cancer-associated fibroblasts [2], and it was found to be an important mechanism during renal and cardiac fibrosis [3, 4]. EndMT was found to be involved in the formation of cerebral cavernous malformations in CCM1 deficient mice [5]
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